To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34(-)c-Kit(+)Sca-1(+)Lin(-) (CD34(-)KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)-HSCs were exclusively present in the Endomucin(+)CD34(-)KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45(-)CD41(+)Endomucin(+) fraction at day 10.5 of gestation (E10.5) and in the CD45(+)CD41(+)Endomucin(+) fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.

Hematopoietic stem cells (HSCs) have been extensively characterized based on functional definitions determined by experimental transplantation into lethally irradiated mice. In mice, HSCs are heterogeneous with regard to self-renewal potential, in vitro colony-forming activity, and in vivo behavior. We attempted prospective isolation of HSC subsets with distinct properties among CD34(-/low) c-Kit+Sca-1+Lin- (CD34-KSL) cells. CD34-KSL cells were divided, based on CD150 expression, into three fractions: CD150high, CD150med, and CD150neg cells. Compared with the other two fractions, CD150high cells were significantly enriched in HSCs, with great self-renewal potential. In vitro colony assays revealed that decreased expression of CD150 was associated with reduced erythroblast/megakaryocyte differentiation potential. All three fractions were regenerated only from CD150high cells in recipient mice. Using single-cell transplantation studies, we found that a fraction of CD150high cells displayed latent and barely detectable myeloid engraftment in primary-recipient mice but progressive and multilineage reconstitution in secondary-recipient mice. These findings highlight the complexity and hierarchy of reconstitution capability, even among HSCs in the most primitive compartment.

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34-/low, c-Kit+, Sca-1+, and lineage marker- (CD34-KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34-KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.

DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34(-/low), c-Kit(+), Sca-1(+), lineage marker(-) (CD34(-) KSL) cells, a fraction of mouse bone marrow cells highly enriched in hematopoietic stem cells (HSCs), expressed both Dnmt3a and Dnmt3b. Using retroviral Cre gene transduction, we conditionally disrupted Dnmt3a, Dnmt3b, or both Dnmt3a and Dnmt3b (Dnmt3a/Dnmt3b) in CD34(-) KSL cells purified from mice in which the functional domains of these genes are flanked by two loxP sites. We found that Dnmt3a and Dnmt3b function as de novo DNA methyltransferases during differentiation of hematopoietic cells. Unexpectedly, in vitro colony assays and in vivo transplantation assays showed that both myeloid and lymphoid lineage differentiation potentials were maintained in Dnmt3a-, Dnmt3b-, and Dnmt3a/Dnmt3b-deficient HSCs. However, Dnmt3a/Dnmt3b-deficient HSCs, but not Dnmt3a- or Dnmt3b-deficient HSCs, were incapable of long-term reconstitution in transplantation assays. These findings establish a critical role for DNA methylation by Dnmt3a and Dnmt3b in HSC self-renewal.